Reference may be made to Journal “Journal of Applied Phycology, 2009, 21: pp 493-507” wherein information available in the literature on Microalgal growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa is described.
Reference may be made to the Report prepared by Tom Bruton for Sustainable Energy Ireland; 2009 (www.sei.ie/algaereport), There are at least 30,000 known species of microalgae which is a very heterogeneous group and not fully explored. From the vast number of known marine and freshwater species, only handfuls are currently of commercial significance. These include Chlorella, Spirulina and Haematococcus. Of these only Dunaliella is predominantly a marine species. Hence, the need is to explore and exploit the Microalgae from marine ecosystem.
Reference may be made to the Journal by Ito et al. “J. Bioscience & Bioengineering, 2005, 100, pp 260-265” wherein the biochemical production of hydrogen and ethanol from the glycerol-containing wastes discharged after biodiesel manufacturing process is described. It is reported that the biochemical activity is much lower than with pure glycerol due to the presence of high salt content in the wastes.
Reference may be made to the patent WO/2008/083352 entitled “Production of biofuels using algae” describing two stage process for production of biofuels from algae including cultivation of an oil-producing algae by promoting sequential photoautotrophic and heterotrophic growth. They co-cultivate nitrogen fixing cyanobacteria to provide nitrogen as nutrient in first stage and subsequently adding sugar obtained from hydrolysis of starch and cellulose. No specific mention is made of the subject matter of the present application.
Reference may be made to the Journal by A. H. Scragg et at “Enzyme and Microbial Technology 2003, 33, pp 884-889” wherein microalgae such as the Chlorella spp. with a cell size in the range of 3-10 μm ideal for combustion in a diesel engine; the liquid fuel consists of an emulsion of biodiesel (transesterified rapeseed oil), a surfactant and cells of Chlorella vulgaris (biomass slurry) used as an unmodified stationary diesel engine for the supply of electricity is described.
Reference may be made to the review paper by Chen (Trends Biotechnology 1996, 14, 421-426) which describes algal oil production and possibility of microalgae to be cultured in heterotrophic conditions where organic carbons, such as sugars and organic acids, serve as carbon sources.
Reference may be made to the paper by Xiaoling Miao et al (Bioresource Technology 2006, 97, pp 841-846) which describes heterotrophically cultivated Chlorella protothecoides (using 10 g/l glucose and 0.1 g/l glycine) to accumulate as much as 55% of its dry weight as oil, compared to only 14% in cells grown photoautotrophically. This patent utilizes costly as well as edible sugars and amino acids like glucose and glycine respectively.
Reference may be made to the patent US0086937A1 by Hazelbeck et al entitled ‘Photosynthetic oil production in a two-stage reactor’ describing two stage reactor for growth and oil production in algae mixing nutrients which contains phosphorous, sulfur, nitrogen, carbonates, numerous trace element with dissolved CO2 and constant agitation involving lot of energy inputs.
Reference may be made to the paper by Han Xu et al (Journal of Biotechnology 2006; 126, pp 499-507) which describes heterotrophic growth of C. protothecoides using corn powder hydrolysate having the crude lipid content of 55.2% in 3L medium in 5L biofermenters. A high density heterotrophic culture of C. protothecoides with CPH feeding was established in the 5 L stirred tank biofermenter. Lipid content in the algal cells cultivated in the biofermenter was 46.1%, which was a little lower than that in the Erlenmeyer flasks (55.3%). The cell growth reached maximum value (3.92 g L−1) after 144 h culture with the substrate of CPH, while the maximum value was 3.74 g L−1 with the substrate of glucose in Erlenmeyer flasks containing 300 mL medium at 28±1° C. under continuous shaking (180 rpm) and air flowing in the dark. It indicated that, it was feasible to use CPH as organic carbon to cultivate Chlorella. 
Reference may be made to the paper by Fu-Ying Feng et al “Process Biochemistry 2005; 40; 1315-1318” wherein effects of glucose, sodium thiosulphate and a combination of these two compounds in culture medium on growth kinetics and fatty acid production of Chlorella sp. Has been described. Two different concentrations (2.5 mmol and 5.0 mmol) of both components in culture medium were used. They suggest that an appropriate concentration of glucose in combination with sodium thiosulphate can enhance the accumulation of lipids of Chlorella sp. cells.
Reference may be made to the paper by Liang, Yanna et al “Biotechnology Letters 2009; 7; 1043-1049”, which describes autotrophic growth with cellular lipid content (38%), and the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l−1) and lipid productivity (54 mg/1/day) was attained using glucose at 1% (w/v) whereas higher concentrations of glucose and glycerol were inhibitory.
Reference may be made to the paper by Chih-Hung Hsieh et al “Bioresource Technology 2009, 100(17), pp 3921-3926” which describes Chlorella sp cultivated in various culture modes to assess biomass and lipid productivity. In the batch mode, the biomass concentrations and lipid content of Chlorella sp. cultivated in a medium containing 0.025-0.200 g L−1 urea were 0.464-2.027 g L−1 and 0.661-0.326 g g−1, respectively. The maximum lipid productivity of 0.124 g L−1 occurred in a medium containing 0.100 g L−1 urea. In the fed-batch cultivation, the highest lipid content was obtained by feeding 0.025 g L−1 of urea during the stationary phase, but the lipid productivity was not significantly increased. However, a semi-continuous process was carried out by harvesting the culture and renewing urea at 0.025 g L−1 each time when the cultivation achieved the early stationary phase. The maximum lipid productivity of 0.139 g d−1 L−1 in the semi-continuous culture was highest in comparison with those in the batch and fed-batch cultivations. Reference may be made to the paper by Mandal et al (Applied Microbiology Biotechnology 2009, 84: 281-291) which describes microalgae such as Scenedesmus obliquus accumulating lipid inside the cell under nitrogen and phosphorous deficient condition. The lipid content increase significantly up to 43% of dry cell weight under N-deficiency.
Reference may be made to the paper by Demirbas “Energy Sources, Part A, 31:163-168, 2009”, which describes comparative lipid profiling of Chlorella protothecoides and Cladophora fracta which contains 29.4% cell dry weight and 14.2% cell dry weight respectively.
Reference may be made to the paper by Cheng et al “Journal of chemical technology & Biotechnology 2009; 84,5; pp 777-781” which describes Chlorella protothecoides utilizing hydrolysate of Jerusalem artichoke tuber (Helianthus tuberosus L) as carbon source and accumulated lipid in vivo, with lipid content as high as 44% cdw, and a carbon source to lipid conversion ratio of about 25% in a 4-day scale cultivation. The lipids were extracted and then converted into biodiesel by transesterification. Cetane acid methyl ester, linoleic acid methyl ester and oleic acid methyl ester were the dominating components of the biodiesel produced. Unsaturated fatty acids methyl ester constituted over 82% of the total biodiesel content.
Reference may be made to the paper by Bertoldi et al “Grasas Y Aceites 2006; 57 (3) pp 270-274” wherein Lipids, fatty acids composition and carotenoids of Chlorella vulgaris cultivated in industrial and agriculture waste waters, the results “showed that lipid contents did not present” significant difference .The use of hydroponic wastewater as an alternative culture medium for the cultivation of Chlorella vulgaris generates good perspectives for lipid, fatty acid and carotenoid production.
Reference may be made to the paper by Xiufeng Li et al “Biotechnology and Bioengineering 2007, 98(4) pp 764-771”, which describes heterotrophic Chlorella protothecoides focused on scaling up fermentation in bioreactors. through substrate feeding and fermentation process controls, the cell density of C. protothecoides achieved 15.5 gL−1 in 5 L, 12.8 gL−1 in 750 L, and 14.2 gL−1 in 11,000 L bioreactors, respectively. Resulted from heterotrophic metabolism, the lipid content reached 46.1%, 48.7%, and 44.3% of cell dry weight in samples from 5 L, 750 L, and 11,000 L bioreactors, respectively.
Reference may be made to the paper by Wei et al “Journal of Industrial Microbiology Biotechnology DOI 10.1007/s10295-009-0624-x”, which describes heterotrophic growth of Chlorella protothecoides using cassava starch hydrolysate i.e. CSH made by two step enzymatic process evolving amylase and gluco-amylase as the organic carbon source, the highest biomass and the maximum total lipid yield obtained were 15.8 and 4.19 g/L, representing increases of 42.3 and 27.7%, respectively, compared to using glucose as the organic carbon source.
It will be evident from the prior art that no cost-effective process has been disclosed for production of Microalgal biomass from biodiesel co-product streams and even with the use of costly co-nutrients and cumbersome 2-step process. The present invention seeks to overcome all the basic limitations and to evolve a novel, simplified and cost-effective process of producing lipids from the microalgal biomass generated from glycerol co-product stream of methyl ester process starting from Jatropha whole seed capsule. Several associated improvements in the process e.g. best utilization of problematic waste, particularly oil sludge generated during mechanical expelling of oil and still bottom of glycerol distillation process, also form part of the present invention, besides involving fed batch process for initially increasing the biomass productivity and then improving the lipid content i.e. lipid IC productivity.